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Functional blockade of v 3 increases total levels and nuclear localization of the CDKs inhibitor p27Kip1 in HRG-overexpressing breast cancer cells. (a) HRG-overexpressing MDA-MB-231 breast cancer cells (231/VEC) and derivatives expressing graded levels of HRG (231/ASPOOL, 231/AS31) were treated with the specific v 3 antagonists S-247 and S-205 for 48 h. Cells were lysed and total protein (20 g) was resolved by SDS–PAGE and then analysed by immunoblot for the CDK inhibitor p27Kip1. Blots were reprobed with an antibody for -actin as a control for protein loading and transfer. The immunoblots were scanned and analysed with the Scion Image software. Densitometric values (columns) provided by the software were plotted as a function of drug concentration. Results are representative of three independent experiments. (b) (Top panel) Immunofluorescent staining for p27Kip1 in 231/VEC and 231/AS31 cells treated with RGD compounds. Cells were seeded in an eight-well chamber slide and treated for 48 h with the v 3 antagonist S-247. Following treatment, cells were incubated with a specific primary antibody against p27Kip1, then with an FITC-conjugated secondary antibody, and the data recorded by epifluorescence microscopy. (Bottom panel) Flow cytometric analysis of p27Kip1 in 231/VEC and 231/AS31 cells after functional blockade of v 3. M1, cells with a positive p27Kip1 fluorescence; GM, Geo Mean fluorescence as provided by the Cell Quest software (Right) p27Kip1 expression index calculated using the formula GM (Geo Mean fluorescence) % of p27Kip1 positive cells. Mean (s.d.) of three independent experiments is plotted (s.d. was less than 1%). (c) (Left panel) Flow cytometric dual analysis of p27Kip1 in the context of DNA content of 231/VEC, 231/ASPOOL and 231/AS31 cells. Bivariate cytograms indicate the levels of p27Kip1 expression in each subcompartment of the cell cycle. (Right panel) Overlapping of the histograms representing p27Kip1 levels in the stated breast cancer models treated with S-247. (Bottom panel) Fold-time increase in the p27Kip1-expression index of 231/VEC, 231/ASPOOL and 231/AS31 cells treated with 0.5 M of v 3 antagonists. p27Kip1 levels were determined separately for each phase of the cell cycle. The data presented summarize the mean (s.d.) of three independent experiments (s.d. was less than 1%)
Source publication
alpha(v)beta(3) integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between alpha(v)beta(3) integrin and overexpression of Heregulin (HRG), a growth factor associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the role of alpha(v)beta(3)...
Contexts in source publication
Context 1
... observed that treatment of MDA-MB-231 cells with increasing concentration of S247 resulted in a solid upregulation of p27 Kip1 levels (1.5-fold increase), as shown in Figure 6a. In contrast, the level of p27 Kip1 in the antisense cells (231/AS POOL and 231/AS31) was slightly changed after the same treatments (Figure 6a). ...
Context 2
... observed that treatment of MDA-MB-231 cells with increasing concentration of S247 resulted in a solid upregulation of p27 Kip1 levels (1.5-fold increase), as shown in Figure 6a. In contrast, the level of p27 Kip1 in the antisense cells (231/AS POOL and 231/AS31) was slightly changed after the same treatments (Figure 6a). In accordance with these results, flow cytometric analysis showed an increase of p27 Kip1 We have previously reported that forced expression of the proangiogenic factor CYR61 into MCF-7 cells increases a v b 3 levels and activates a v b 3 -driven cell signaling ( Menendez et al., 2004 ...
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Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-indu...
Citations
... A number of integrins are found to have significant differences in expression levels between tumor and normal tissues and hence provide potential targets for cancer therapy. Among various integrins, the a V b 3 integrin has significantly upregulated expression level in cancers such as melanoma, breast cancer, prostate cancer, pancreatic cancer, ovarian cancer, cervical cancer, and glioblastoma compared to that in normal epithelial cells and is thus identified as an effective marker and drug target for cancer (67,68). Based on the fact that integrin a V b 3 can specifically bind to the Arg-Gly-Asp (RGD) motif present in various extracellular matrix (ECM) ligands, researchers have developed several targeted drugs carrying RGD motif, among which cyclic RGD peptide Cilengitide has shown promising prospects in clinical trials targeting a range of cancers including lung cancer, prostate cancer, and glioblastoma (66,69,70). ...
Extracellular vesicles (EVs), characterized by low immunogenicity, high biocompatibility and targeting specificity along with excellent blood-brain barrier permeability, are increasingly recognized as promising drug delivery vehicles for treating a variety of diseases, such as cancer, inflammation and viral infection. However, recent findings demonstrate that the intracellular delivery efficiency of EVs fall short of expectations due to phagocytic clearance mediated by the host mononuclear phagocyte system through Fcγ receptors, complement receptors as well as non-opsonic phagocytic receptors. In this text, we investigate a range of bacterial virulence proteins that antagonize host phagocytic machinery, aiming to explore their potential in engineering EVs to counteract phagocytosis. Special emphasis is placed on IdeS secreted by Group A Streptococcus and ImpA secreted by Pseudomonas aeruginosa, as they not only counteract phagocytosis but also bind to highly upregulated surface biomarkers αVβ3 on cancer cells or cleave the tumor growth and metastasis-promoting factor CD44, respectively. This suggests that bacterial anti-phagocytic proteins, after decorated onto EVs using pre-loading or post-loading strategies, can not only improve EV-based drug delivery efficiency by evading host phagocytosis and thus achieve better therapeutic outcomes but also further enable an innovative synergistic EV-based cancer therapy approach by integrating both phagocytosis antagonism and cancer targeting or deactivation.
... In addition to acting as an oncogene in BC, ovarian cancer, stomach cancer, pancreatic cancer, and glioblastoma (18,19), CYR61 also serves as a tumor suppressor in human hepatocellular carcinoma and non-small cell lung cancer (20)(21)(22)(23)(24). CYR61 physically contributing to cardiovascular development throughout embryogenesis (13). Proliferation, survival, and angiogenesis are induced by its binding to integrin αvβ3 (13,25,26). Additionally, it has been hypothesized that CYR61 stimulates angiogenesis by increasing the production of vascular endothelial growth factors (27,28). ...
Background/aim:
Hormone sensitivity-targeted therapy with selective estrogen receptor modulators (SERMs), such as 4-hydroxytamoxifen (4-OHT), is the mainstay of treatment for breast cancers (BCs) that express estrogen receptor α (ERα). However, development of resistance limits this therapy approach. The question arises whether changes associated with 4-OHT resistance could be exploited therapeutically.
Materials and methods:
First, 4-OHT-resistant sublines of ERα-positive breast carcinoma cell lines MCF-7 and T47D were generated. Viability was assessed by the Alamar Blue assay. Cell invasion was quantified in modified Boyden chambers with Matrigel. Changes in expression of CYR61, S100A4, and ERα were examined by RT-qPCR. Expression of CYR61 was suppressed by transient gene silencing using siRNA. Successful suppression was verified by western blot. Efficacy of 4-OHT treatment was analyzed by quantification of viability using Alamar Blue assay. Correlation of CYR61 levels in patients with luminal A BC to distant metastases-free survival was determined by Kaplan-Meier analysis.
Results:
ERα-positive MCF-7 and T47D BC cells exhibit an extremely weak invasion rate. Acquired tamoxifen resistance significantly increased the invasive behavior of both tamoxifen-resistant MCF-7-TR and T47D-TR sublines. In addition, expression of CYR61 and S100A4 showed significantly increased levels, whereas expression of ERα was decreased. Suppression of CYR61 expression resulted in a significant decreased invasion rate. In addition, expression of S100A4 was reduced, whereas expression of ERα was increased. Furthermore, suppression of CYR61 resulted in re-sensitization to 4-OHT. High CYR61 levels in patients with luminal A BC resulted in reduced distant metastases-free survival.
Conclusion:
The prometastatic factor CYR61 appears to play an important role in the increased invasiveness of tamoxifen-resistant ERα-positive BC cells. Its suppression leads to a lower invasion rate. Given the few therapeutic options available for tamoxifen-resistant BC, therapy that reduces CYR61 may improve its treatability in future.
... As for the negative control groups NPs2 and NPs3, red fluorescence appeared in the cell cytoplasm rather than staying at the cell surface (Figure 2(a)), and their Pearson correlation coefficients were þ0.18 and þ0.17, respectively, indicating that NPs2 or NPs3 and avb6 protein had no colocalization (Supplementary Figure S5). MDA-MB-231 and HepG2 cells which rarely expressed avb6 protein were also treated with NPs1 for 8 h, respectively (Vellon et al., 2005;Patsenker et al., 2010). As expected, NPs1 did not stay on these cell surfaces in the absence of avb6 protein (Supplementary Figure S6). ...
Photodynamic therapy (PDT) and photothermal therapy (PTT) have attracted research interest for their noninvasive nature and selective treatment of tumor tissues. They are effective through the generation of reactive oxygen species (ROS) or heat. Nevertheless, several problems, including low bioavailability and long-lasting cutaneous photosensitivity, have limited their clinical application. In this study, we reported an in situ self-assembly strategy that could improve various biological properties of the photosensitizer in vivo. A photosensitizer connected to a receptor-mediated smart peptide can self-assemble into nanoparticles (NPs) under the force of hydrophobic interaction and then transform into a nanofibrillar network after attaching to the tumor cell surface with the help of the β-sheet-forming peptide KLVFF. The supramolecular structural changes deeply affected the PDT and PTT properties of the photosensitizer on tumors. After being aggregated into the nanostructure, the water solubility and targeting ability of the photosensitizer was ameliorated. Moreover, the improvement of the photothermal conversion efficiency, ROS generation, and tumor retention followed the formation of nanofibrils (NFs). This self-assembly strategy showed the ability of supramolecular nanofibrils to improve the bioavailability of photosensitizers, which provides a new potential treatment avenue for various cancer therapies.
... CCN1 depletion leads to the suppression of α v β 3 overexpression, thereby generating a dominant negative phenotype capable of impairing the bioactivity of other growth factors that participate in the high proliferative and migratory capacity of triple-negative/basal-like BC cells. The ability of CCN1 to activate MAPK signaling downstream of α v β 3 is likely to be involved in the anti-proliferative and antimigratory effects of CCN1 silencing in triplenegative/basal-like BC cells [27,39,40]. CCN1 depletion also completely blocks the ability of triple-negative/basal-like BC cells to proliferate and form colonies in semi-solid agarose gel. ...
Triple-negative/basal-like breast cancer (BC) is characterized by aggressive biological features, which allow relapse and metastatic spread to occur more frequently than in hormone receptor-positive (luminal) subtypes. The molecular complexity of triple-negative/basal-like BC poses major challenges for the implementation of targeted therapies, and chemotherapy remains the standard approach at all stages. The matricellular protein cysteine-rich angiogenic inducer 61 (CCN1/CYR61) is associated with aggressive metastatic phenotypes and poor prognosis in BC, but it is unclear whether anti-CCN1 approaches can be successfully applied in triple-negative/basal-like BC. Herein, we first characterized the prevalence of CNN1 expression in matched samples of primary tumors and metastatic relapse in a series of patients with BC. We then investigated the biological effect of CCN1 depletion on tumorigenic traits in vitro and in vivo using archetypal TNBC cell lines. Immunohistochemical analyses of tissue microarrays revealed a significant increase of the highest CCN1 score in recurrent tissues of triple-negative/basal-like BC tumors. Stable silencing of CCN1 in triple-negative/basal-like BC cells promoted a marked reduction in the expression of the CCN1 integrin receptor αvβ3, inhibited anchorage-dependent cell growth, reduced clonogenicity, and impaired migration capacity. In an orthotopic model of triple-negative/basal-like BC, silencing of CCN1 notably reduced tumor burden, which was accompanied by decreased microvessel density and concurrent induction of the luminal epithelial marker E-cadherin. Thus, CNN1/CYR61-targeting strategies might have therapeutic value in suppressing the biological aggressiveness of triple-negative/basal-like BC.
... Our own previous studies and those of others have established a significant correlation between elevated levels of CCN1 and more advanced disease and metastatic phenotypes in in vitro breast cancer models and in patients [14-17, 20, 29-33]. Specifically, we demonstrated that the ability of CCN1 to drive breast tumor initiation, vascularization, and invasiveness, as well as to provide protection of breast cancer cells against chemotherapy-induced apoptosis, was largely mediated through binding to integrin αvβ3, whose expression is also induced by CCN1 [30,34,35]. In vitro studies have also clarified the ability of CCN1 to overcome estrogen dependency and elicit resistance to the selective estrogen receptor (ER) modulators and down-regulators (SERMs/SERDs) tamoxifen and fulvestrant in ER-positive breast cancer cells [15-17, 29, 31]. ...
CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor αvβ3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α6β1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either αvβ3 or α6β1. First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to αvβ3 and α6β1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to αvβ3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α6β1, failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α6β1-binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α6β1, but not via αvβ3, drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.
... [268] Integrins continue to regulate cell growth in some tumors, although adhesion-dependent control of proliferation is normally deregulated. [275,276] On the surface of tumor-associated host cells, they can decisively influence the malignant potential of a tumor. Their role in cell migration and invasion is one of the most studied functions in tumor biology. ...
Chimeric antigen receptors (CARs) are able to specifically direct T cells to tumor antigens and therapy with anti-CD19 CARs has already cured cancer patients with B-cell lymphomas who have undergone long-term therapy non-successful. Despite this impressive result, the therapy is currently only approved as a last treatment option for blood cancers due to its life-threatening deficiencies. For patient safety and to enable additional application such as the treatment of solid tumors, CAR-T cells must be controllable, e. g. by chemically programmable CARs (cpCARs) regulated by hapten-like compounds. This thesis reports the synthesis and characterization of such hapten-like compounds. In the first step, seven different warheads with two different spacers were bound to biotin in order to find a suitable warhead for programming the cpCAR. In a second step, synthetic routes for the three pharmacophores folate, c(RGD), and an RGD peptidomimetic were developed. The routes allow the modification of the pharmacophores with one of the warheads from the first step. CuAAC was chosen as a bioorthogonal approach to link pharmacophores and warheads. In total, three different pharmacophores were modified with the 1,3-diketone motif of compound 21 leading to 112, 113 and 128. Activation of the T-cell signaling cascade was tested after binding of these hapten-like compounds to the cpCAR in the presence of suitable target structures. For 112, only a slight, non-significant, activation of the T-cell signaling cascade was observed, whereas for 113 and 128, a significant activation of the T-cell signaling cascade was observed. The poor solubility of the folate compounds led to alternative strategies. Folic acid was exchanged by pteroic acid and the bifunctional, linear compounds were enlarged to trifunctional dendrimers. Besides the reported regioisomer in 112, a second one, which was not reported to date, occurred by the cyclization of the linear RGD pentapeptide leading to 113. After the reported synthesis of an RGD peptidomimetic analogous to 128 could not be reproduced, a new synthetic route was developed. It also consists of 17 steps, but reduces the number of linear steps from 13 to 10. Moreover, the developed route contains an asymmetric hydrogenation step and is, compared to the published one, more flexible by the use of the copper-catalyzed azide-alkyne cycloaddition (CuAAC). In addition, an unknown reaction was observed. Instead of the formation of a Schiff base in the reductive amination of 129, an insertion of propargylamine occurred forming 131. The reaction is almost quantitative and in high purity. After requiring no purification, it could be predestined for industrial purposes, such as the synthesis of N-functionalized 1,2-dihydroquinolines or as a building block with various orthogonal functional groups. Besides the sulfonamide 16, the diketone (21, 27, 31) and lactam compounds (39 – 41), experiments on adapter molecules with further warheads were performed. In the synthesis of a proadapter approach, in which the warhead is formed only after the retro-aldol reaction catalyzed by the mAb, 6 of 10 steps were successfully performed. A newly developed synthesis to keto-sulfonyl and keto-sulfoxide compounds could not be completed but was performed on a small scale to the point of keto-sulfonyl and keto-sulfoxide. Furthermore, a universal synthesis route was designed to allow the introduction of the warhead at the end of the synthesis by acylation. Thus, after 5 shared steps, 3 of them in quantitative yield, different warheads may be introduced. Moreover, this also facilitates the purification and the analysis of the compounds by the absence of tautomerism or labile groups. However, the acylation experiments were not successful with either the acid cyanide or the Weinreb amide. In summary, this thesis has proven that the 1,3-diketone motif is a suitable warhead for programming the cpCAR, which was developed by Hudecek et al. (unpublished data). The hapten-like compounds 112, 113 and 128 simultaneously bind to integrin ${\alpha}_v{\beta}_3$ and the cpCAR activating the T-cell signaling cascade. The modular synthesis strategy and the use of the bioorthogonal CuAAC allow straightforward access to these valuable immunotherapeutics but revealed the need for an additional purification step to remove copper ions.
... Rac recruits activated integrins to the leading edge to promote cell migration [32]. Among these is the integrin αvβ3, whose expression is in turn stimulated by CCN1 [33]. By inhibiting CCN1, integrin αvβ3 will not be activated and therefore, will not be recruited by Rac to stimulate the positive feedback loop and promote elongated cell migration. ...
Cancer cell invasion is a precondition for tumour metastasis and represents one of the most devastating characteristics of cancer. The development of drugs targeting cell migration, known as migrastatics, may improve the treatment of highly invasive tumours such as glioblastoma (GBM). In this study, investigations into the role of the cell adhesion protein Cellular communication network factor 1 (CCN1, also known as CYR61) in GBM cell migration uncovered a drug resistance mechanism adopted by cells when treated with the small molecule inhibitor CCG-1423. This inhibitor binds to importin α/β inhibiting the nuclear translocation of the transcriptional co-activator MKL1, thus preventing downstream effects including migration. Despite this reported role as an inhibitor of cell migration, we found that CCG-1423 treatment did not inhibit GBM cell migration. However, we could observe cells now migrating by mesenchymal–amoeboid transition (MAT). Furthermore, we present evidence that CCN1 plays a critical role in the progression of GBM with increased expression in higher-grade tumours and matched blood samples. These findings support a potential role for CCN1 as a biomarker for the monitoring and potentially early prediction of GBM recurrence, therefore as such could help to improve treatment of and increase survival rates of this devastating disease.
... It regulates several signaling pathways, such as NRG1-ERBB, FGF1, and IGF1 signaling [193][194][195]. Therefore, it can be introduced as a pivotal regulator of angiogenesis, adhesion, and metastasis [196]. There was an inverse association between levels of miR-338 and ITGB3 expression in lung tumor tissues [197]. ...
Background
Cancer, as one of the main causes of human deaths, is currently a significant global health challenge. Since the majority of cancer-related deaths are associated with late diagnosis, it is necessary to develop minimally invasive early detection markers to manage and reduce mortality rates. MicroRNAs (miRNAs), as highly conserved non-coding RNAs, target the specific mRNAs which are involved in regulation of various fundamental cellular processes such as cell proliferation, death, and signaling pathways. MiRNAs can also be regulated by long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). They are highly stable in body fluids and have tumor-specific expression profiles, which suggest their suitability as efficient non-invasive diagnostic and prognostic tumor markers. Aberrant expression of miR-338 has been widely reported in different cancers. It regulates cell proliferation, migration, angiogenesis, and apoptosis in tumor cells.
Main body
In the present review, we have summarized all miR-338 interactions with other non-coding RNAs (ncRNAs) and associated signaling pathways to clarify the role of miR-338 during tumor progression.
Conclusions
It was concluded that miR-338 mainly functions as a tumor suppressor in different cancers. There were also significant associations between miR-338 and other ncRNAs in tumor cells. Moreover, miR-338 has a pivotal role during tumor progression using the regulation of WNT, MAPK, and PI3K/AKT signaling pathways. This review highlights miR-338 as a pivotal ncRNA in biology of tumor cells.
... In addition, several tumor microenvironment (TME) components, including biological molecules such as growth factors and proteases owe their localization to certain regions to integrins (Dzobo et al., 2012(Dzobo et al., , 2014. Thus, integrins can influence cellular prolifer-ation and signaling through ''capturing'' growth factors and proteases in certain regions of the TME (Assoian and Klein, 2008;Vellon et al., 2005). ...
... New findings show that specific integrins are required by certain growth factors and oncogenes during tumor initiation and growth (Assoian and Klein, 2008;Vellon et al., 2005). This makes it critical to delineate the crosstalk between integrins and growth factors and oncogenes during drug development. ...
Many cellular functions important for solid tumor initiation and progression are mediated by members of the integrin family, a diverse family of cell attachment receptors. With recent studies emphasizing the role of the tumor microenvironment (TME) in tumor initiation and progression, it is not surprising that considerable attention is being paid to integrins. Several integrin antagonists are under clinical trials, with many demonstrating promising activity in patients with different cancers. A deeper knowledge of the functions of integrins within the TME is still required and might lead to better inhibitors being discovered. Integrin expression is commonly dysregulated in many tumors with integrins playing key roles in signaling as well as promotion of tumor cell invasion and migration. Integrins also play a major role in adhesion of circulating tumor cells to new sites and the resulting formation of secondary tumors. Furthermore, integrins have demonstrated the ability to promoting stem cell-like properties in tumor cells as well as drug resistance. Anti-integrin therapies rely heavily on the doses or concentrations used as these determine whether the drugs act as antagonists or as integrin agonists. This expert review offers the latest synthesis in terms of the current knowledge of integrins functions within the TME and as potential molecular targets for cancer therapeutics innovation.
... CYR61 plays a role in multiple physiological functions, including development, tissue repair, cell adhesion, migration, and proliferation (22). Previous studies have shown CYR61 overexpression is associated with invasion and poor prognosis in several types of cancer, including breast cancer (23,24), gastric cancer (25,26), glioblastoma multiforme (27,28), and oral cancer (29,30). However, the correlation between CYR61 and BC is largely unknown. ...
... A significant increase in the urinary CYR61 level in MIBCs was observed compared with NMIBCs; urinary CYR61 was significantly higher in BC patients than controls without BC (P=0.007) ( Figure S3). The prognostic value of CYR61 overexpression in tissue has been previously studied in breast cancer (23,24), gastric cancer (25,26), glioblastoma multiforme (27,28), oral cancer (29,30), and ovarian epithelial carcinoma (34). However, as a soluble diagnostic or prognostic biomarker, CYR61 has been only studied in acute coronary syndrome and systemic lupus erythematosus (35). ...
Background:
The biological behaviors, clinical treatment, prognosis of non-muscle-invasive bladder cancers (NMIBCs) and muscle-invasive bladder cancers (MIBCs) are distinct. Accurate staging is pivotal in optimal therapy planning for bladder cancers (BCs). However, it is insufficient for urologists in preoperative determining whether the tumor has invaded within the muscularis propria through cystoscope and imaging methods (CT or MRI). Therefore, searching for ideal biomarkers from the tumor tissues and urine is important for identifying the MIBCs preoperatively.
Methods:
Differentially expressed genes between NMIBCs and MIBCs were identified by microarray analysis and validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemical analysis. The correlation between cysteine-rich angiogenic inducer 61 (CYR61) expression and Kaplan-Meier test evaluated patients' overall survival (OS). CYR61 protein levels were measured using enzyme-linked immunosorbent assay (ELISA) in preoperatively collected urine samples from BC patients. The receiver-operating characteristic (ROC) curve analyzed the diagnostic accuracy of uric CYR61. The siRNA mediated silencing of CYR61 in bladder carcinoma cells was performed using Lipofectamine 2000. Cell migration and invasion were assessed using wound healing and transwell assay, respectively.
Results:
Differential gene expression analysis using microarray between 14 MIBCs and 16 NMIBCs human tumor samples revealed a significant increase (P<0.001) in the expression of CYR61 in MIBCs compared with NMIBCs. Higher expression of CYR61 in MIBCs was found in additional 54 tumor samples using qRT-PCR. Therefore, the overexpression of CYR61 in MIBCs could be used as a potential biomarker to distinguish between MIBCs and NMIBCs. ELISA detected elevated levels of CYR61 in the urine samples of MIBC patients (average 2.5-fold) compared with NMIBCs, with 72.7% sensitivity and 86.0% specificity to distinguish MIBCs from NMIBCs. Wound healing and transwell assays using CYR61-silenced carcinoma cells indicated the role of CYR61 in cell migration and invasion.
Conclusions:
CYR61 expression is higher in MIBCs compared with NMIBCs and can serve as a promising biomarker for the preoperative diagnosis of MIBCs with prognostic value; however, multicentric prospective validation is essential for the further evaluation of CYR61.
